Cancer patients can harbor high numbers of activated tumor-specific T cells that may exhibit tumoricidal activity in vitro but in most cases fail to eradicate the tumor in vivo. Understanding the reasons for the failure of anti-tumor immune responses, as well as developing efficient therapeutic approaches, requires the elucidation of in vivo mechanisms behind T cell responses both in lymphoid and nonlymphoid tissues. The study of immune responses has been largely limited to cells retrieved from lymphoid tissues that are analyzed either ex vivo or upon in vitro restimulation. The overall goal of this Project is to validate and employ noninvasive intravital imaging techniques to objectively monitor spontaneously arising tumor-specific T cell responses as well as measure the impact that immunotherapeutic modalities have on these responses in vivo. In the first aim, we will study how tumor-specific CD4 T cells (Theiper and Treg) interfere with the migration of tumor-specific CD8 T cells to tumors. In the second aim, we will use novel multimodal cell trackers with potential use in humans for the monitoring of in vitro activated tumor-specific T cells following adoptive transfer immunotherapy in tumor-bearing mice. In the third aim, we will determine the interactive and cytotoxic activity of tumor-specific CDS T cells in tumor draining lymph nodes and in the tumor bed. There is a strong emphasis on deciphering how tumor-specific CD4 Treg control anti-tumor immune responses, and how their interference can be prevented by using efficient immunotherapies. The project will interact closely with the Ntziachristos lab (Lab for Biooptics and Molecular Imaging at CMIR) in further developing and validating fluorescent protein tomography, and with several immunology groups at Harvard (von Boehmer and Khazaie at DFCI, Mathis at Joslin). The ultimate goal of this project is to understand how immune tolerance is controlled in vivo in order to develop more effective immunotherapies.